The sphingolipid metabolite sphingosine-1 phosphate (SPP) is thought to be a second messenger for regulating mammalian cell growth and other processes. However, the physiological significance of SPP signaling has yet to be established. Likewise, we have only a rudimentary understanding of how SPP synthesis and turnover are regulated and what signal transduction pathways it controls. To begin to establish the physiological function(s) of long chain base (LCB) phosphates like SPP and to understand how their synthesis is regulated, we propose to study LCB phosphates in Saccharomyces cerevisiae. Selection techniques enabled us to isolate two genes, LCB4 and LCB5, that encode LCB kinases, the first genes of this type to be isolated. The homologous Lcb4 and Lcb5 proteins are not related to any in known protein, thus revealing a new class of lipid kinase. The Lcb4 and Lcb5 proteins account for 95% and 5%, respectively, of the Lcb kinase activity in yeast cells. About half of the Lcb4 protein is in the membrane fraction of the cell, which presents an enigma because the predicted protein has no membrane-binding domain. This proposal seeks to determine how LCB phosphate synthesis is regulated and what the functions of the LCB phosphates are. Specific Aim 1 is to purify and characterize the enzymatic proteins of the Lcb4 and Lcb5 kinases. Specific Aim 2 is to determine whether the two proteins are located in cells and if their location changes in a regulated manner. Specific Aim 3 is to locate domains in the two proteins for substrate binding, catalysis, and protein localization. Specific Aim 4 is to determine which cellular processes require LCB phosphates for function. The results of these studies will form a foundation for understanding the function of this ubiquitous class of lipid phosphates in mammals, including their proposed roles in growth regulation, apoptosis, regulation of heart rate, activation of platelets and mast cells, and neurite retraction.